The focus of this work has been to understand the molecular details that control the interaction of MHC molecules with NK receptors, T cell receptors, and with coreceptors such as CD8. These studies are dependent upon both functional and biophysical analysis of the interaction of the molecules in question, and attempts are made to correlate binding properties with function and structure. Our most exciting progress in the past several years has been the development of a general model system for both the engineering of NK receptors of the C-type lectin family, and the use of these in biophysical binding studies. These experiments are of significant interest and importance to a basic understanding of immune recognition because they allow the characterization of the molecular details of a family of cell-cell interactions that reflect the innate rather than the adaptive immune system. We have engineered and isolated NK receptors, Ly49A, Ly49C, and Ly49G2, representatives of the inhibitory receptors of the C-type lectin family. We have shownthat Ly49A, which is produced as a non-covalent dimer, binds H-2Dd in the absence of Ca++ (indicating that this is not a bona fide C-type lectin). It binds bacterial H-2Dd (which lacks carbohydrate) indicating that carbohydrate is not required for the interaction. We have characterized the lack of peptide specificity of the interaction, and using both analytical ultracentrifugation and surface plasmon resonance we have been able to measure the affinity of interaction of Ly49A with H-2Dd. The ability to bind H-2Dd resides in a fragment of the Ly49A molecule that extends from amino acid 127 to the terminus at 262, the fragment that we have crystallized. The affinity of the Ly49A for H-2Dd as determined at 25 degrees C by surface plasmon resonance in many determinations ranges from 6.1 to 26 micromolar, and is characterized by association rate constant ka of 1300 to 5100 M-1s-1 and dissociation rate constant kd of 0.024 to 0.031 s-1. These parameters are remarkably similar to those that we have observed in the binding of H-2Dd to a cognate T cell receptor. We have demonstrated the ability of H-2Dd to simultaneously interact with both the cognate T cell receptor and the Ly49A NK receptor. This has important biological implications in that a number of bona fide T cells have been identified as expressing both T cell receptors as well as NK receptors. If a T cell bearing an inhibitory or activating NK receptor can bind the same MHC molecule simultaneously, there is the opportunity for complex regulatory signaling based on these interactions. Additional experiments are underway characteirzing the details of the interaction Ly49A with H-2Dd, using in vitro mutants of both Ly49A and H-2Dd. Mutants of Ly49A confirm the view that there are specific regions of the molecule that interact with H-2Dd and other regions that are globally critical for its overall structure. Mutants of H-2Dd suggest that the MHC molecule interacts over a wide surface area with the NK receptor ligand.